B. Research Project/Resource Production Summary Transcription factors (TFs) are proteins that contain site-specific DNA binding domains and regulate gene expression during development. The complete set of TFs are known for C. elegans and D. melanogaster and with their small genome sizes and well studied embryonic development, it is possible to determine their in vivo binding profiles and function. The modENCODE and modERN projects have generated ChIP-seq binding profiles for a substantial number of TFs. Our output has dramatically increased during modERN through the use of transgenic organisms expressing tagged-TFs. This approach has proven to be more practical than developing TF-specific antibodies, and has been adopted by other projects such as ENCODE. These transgenic lines constitute a valuable resource to the fly and worm communities. We aim to continue our production of GFP-tagged TFs, producing lines for the remaining 340 TFs in worm and 260 in flies. Furthermore, we will continue to use these new lines along with recently generated lines to generate ChIP-seq data, generating binding profiles for all expressed TFs in both organisms, ~400 in each. During the modERN project we created a pipeline to knockdown TF expression using RNAi and identified the genes that showed differential expression using RNA-seq. While we have had success knocking-down TFs with RNAi in Drosophila, in C. elegans we have utilized homozygous viable deletion strains. In both, we assay expression in embryos before the onset of TF expression, at the peak of expression, and two hours post peak or at terminal differentiation. Using these two approaches, we will continue to generate RNA-seq data on TF loss-of-function lines, focusing on TFs with orthologs in humans or between fly and worm.